PHYTOESTROGEN CONTENTS OF SELECTED FOODS ESRA GÜLTEKN - PDF

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PHYTOESTROGEN CONTENTS OF SELECTED FOODS ESRA GÜLTEKN SEPTEMBER 24 PHYTOESTROGEN CONTENTS OF SELECTED FOODS A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF THE MIDDLE EAST

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PHYTOESTROGEN CONTENTS OF SELECTED FOODS ESRA GÜLTEKN SEPTEMBER 24 PHYTOESTROGEN CONTENTS OF SELECTED FOODS A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF THE MIDDLE EAST TECHNICAL UNIVERSITY BY ESRA GÜLTEKN IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF FOOD ENGINEERING SEPTEMBER 24 Approval of the Graduate School of Natural and Applied Sciences Prof. Dr. Canan Özgen Director I certify that this thesis satisfies all the requirements as a thesis for the degree of Master of Science. Prof. Dr. Levent Bayındırlı Head of Department This is to certify that we have read this thesis and that in our opinion it is fully adequate, in scope and quality, as a thesis for the degree of Master of Science. Prof. Dr. Fatih Yıldız Supervisor Examining Committee Members Prof. Dr. Ufuk Gündüz (METU, BIO) Prof. Dr. Fatih Yıldız (METU, FDE) Prof. Dr. Sedat Veliolu (AU, FDE) Assoc. Prof. Dr. Esra Yener (METU, FDE) Assoc. Prof. Dr. Fatih zgü (METU, BIO) I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last Name : Signature : iii ABSTRACT PHYTOESTROGEN CONTENTS OF SELECTED FOODS Gültekin, Esra M. Sc., Department of Food Engineering Supervisor : Prof. Dr. Fatih Yıldız September 24, 46 pages Phytoestrogens are naturally occurring chemicals of plant origin that have the ability to cause estrogenic and/or anti-estrogenic effects due to their structural similarities to the human hormone oestradiol. It has been proposed that phytoestrogens protect against a wide range of ailments, including breast and prostate cancers, cardiovascular disease, osteoporosis, and menopausal symptoms. Daidzein, biochanin A and especially genistein which has been reported to be the most biologically active dietary phytoestrogen attract great deal of interest in today s researches. iv In this study, twenty different food items, including legumes, fruits, nuts and herbs, (haricot beans, chickpeas, green lentils, red lentils, soybeans, licorice root, yarrow, dried chestnuts, prunes, raisins, currants, black cumin, dried apricots, dried parsley, dried dates, dried figs, sage (from Aegean region), sage (from Mediterranean region), grapevine leaves, gilaburu) were selected. Following an extraction procedure employing acid hydrolysis and heating; they were analysed for their daidzein, genistein and biochanin A contents using a reversed-phase C 18 column with linear gradient elution on a high-performance liquid chromatography (HPLC) coupled with diode-array detector (DAD). Soybeans were found to contain high amounts of daidzein (91.36 mg/1 g) and genistein (85.57 mg/1 g). Chickpeas were found to contain much less amount of genistein (.89 mg/1 g) compared with that of soybeans and also biochanin A (.95 mg/1 g) which was not detected in soybeans. None of daidzein, genistein and biochanin A was detected in the remaining eighteen food items. Keywords : Phytoestrogen; daidzein; genistein; biochanin A; HPLC-DAD v ÖZ SEÇLM GIDALARIN FTOÖSTROJEN ÇERKLER Gültekin, Esra Yüksek Lisans, Gıda Mühendislii Bölümü Tez Yöneticisi : Prof. Dr. Fatih Yıldız Eylül 24, 46 sayfa Fitoöstrojenler; insan hormonu östradiole yapısal benzerlikleri dolayısıyla östrojenik ve/veya anti-östrojenik etkiler gösteren, bitki kökenli doal kimyasal bileiklerdir. Fitoöstrojenlerin; göüs ve prostat kanseri, kalp-damar hastalıkları, osteoporoz ve menopoz belirtileri dahil pekçok hastalıa karı koruyucu etkisi bulunduu düünülmektedir. Daidzein, biochanin A ve özellikle de biyolojik olarak en aktif fitoöstrojen olduu belirtilen genistein, günümüz aratırmalarında büyük ilgi çekmektedir. vi Bu çalımada; yirmi farklı gıda maddesi (kuru fasulye, nohut, yeil mercimek, kırmızı mercimek, soya fasulyesi, meyankökü, civanperçemi, kuru kestane, kuru erik, kuru üzüm, ku üzümü, çörekotu, kuru kayısı, kuru maydonoz, kuru hurma, kuru incir, adaçayı (Ege Bölgesi), adaçayı (Akdeniz Bölgesi), asma yapraı, gilaburu) belirlenmitir. Asit hidrolizi ve ısıtma aamalarını içeren bir ekstraksiyon prosedürünü takiben; yüksek performanslı sıvı kromatografisi (HPLC) ve diyot-array dedektör (DAD) kullanılarak, ters-fazlı C 18 kolonda, lineer gradient yöntemle, bu gıdaların daidzein, genistein ve biochanin A içerikleri aratırılmıtır. Soya fasülyesinin yüksek miktarlarda daidzein (91.36 mg/1 g) ve genistein (85.57 mg/1 g) içerdii saptanmıtır. Nohutta, soya fasulyesindeki genistein miktarından çok daha düük miktarda genistein (.89 mg/1 g) ve ayrıca soya fasulyesinde tespit edilmeyen biochanin A (.95 mg/1 g) saptanmıtır. Dier onsekiz gıdada ise, daidzein, genistein ve biochanin A bileiklerinin hiçbiri tespit edilmemitir. Anahtar Kelimeler : Fitoöstrojen; daidzein; genistein; biochanin A; HPLC-DAD vii To My Family and Gürkan viii ACKNOWLEDGMENTS The author wishes to express her deepest gratitude to her supervisor, Prof. Dr. Fatih Yıldız, for his guidance, advice and insight throughout the research. The author is indepted to Prof. Dr. Nevzat Artık for his understanding and very important contribution of arranging Ankara Provincial Control Laboratory of Ministry of Agricultural and Rural Affairs, Prof. Dr. Sedat Veliolu for his criticism and comments, Dr. Neslihan Alper for her being so helpful and so informed, Dr. Ender Sinan Poyrazolu for the considerably important time he spent, Dr. Zeliha Yıldırım for her kindness, Nihal Türkmen for her encouragement, Nurten Çankaya and Ferda Elmalıpınar for their interest. Food Engineering Laboratory in Ankara University and Ankara Provincial Control Laboratory, where the experiments were carried out, are gratefully appreciated. The suggestions and comments of Dr. Aytanga Ökmen and the technical assistance of Aytekin Güler, Elif Doan and Fatma Cokun are gratefully acknowledged. The author also wishes to express her deepest feelings to her mother, father, little brother for their endless support, and would like to extent warm thanks especially to her friend Gürkan for his understanding, support and help. This study was supported by the Scientific and Technical Research Council of Turkey (TÜBTAK) (TOGTAG 3179). ix TABLE OF CONTENTS PLAGIARISM...iii ABSTRACT...iv ÖZ...vi ACKNOWLEDGMENTS...ix TABLE OF CONTENTS...x LIST OF TABLES... xii LIST OF FIGURES.....xiii LIST OF ABBREVIATIONS...xv CHAPTER 1. INTRODUCTION Phytoestrogens Classes of Phytoestrogens Structural Similarities of Phytoestrogens to Oestradiol Oestrogenic Properties of Phytoestrogens Oestrogenic Potencies of Phytoestrogens Isoflavones Classes of Isoflavones Physical and Chemical Properties of Isoflavones Water Solubility Chemical Stability Absorption, Distribution, Metabolism and Excretion of Isoflavones...7 x 1.3. Analysis of Phytoestrogens Isolation of Phytoestrogens Analytical Methods Reported Phytoestrogen Contents of Foods MATERIALS AND METHODS Materials Food Materials Standards Solvents and Reagents Methods Experimental Plan Preparation of Samples Preparation of Standard Solution HPLC-DAD Analysis RESULTS AND DISCUSSIONS Results for Standard Mixture Chromatogram of Standard Mixture Determination of the Proper Wavelength Retention Times of the Analytes Screening of Twenty Samples Evaluation of Chromatograms of Twenty Samples Qualitative Determination of Daidzein and Genistein in Soybeans Qualitative Determination of Genistein and Biochanin A in Chickpeas Quantitative Determination of Daidzein and Genistein in Soybeans Quantitative Determination of Genistein and Biochanin A in Chickpeas Phytoestrogen Contents of Twenty Different Food Materials Evaluation of the Results CONCLUSIONS AND RECOMMENDATIONS REFERENCES...4 APPENDICES 46 xi LIST OF TABLES TABLES 1.1 Key structural elements crucial for estradiol-like action Daidzein, genistein and biochanin A contents of soybeans Daidzein, genistein and biochanin A contents of chickpeas Daidzein, genistein and biochanin A contents of lentils Daidzein, genistein and biochanin A contents of beans Daidzein and genistein contents of fruits and nuts Food materials analysed Absorbance maxima of daidzein, genistein and biochanin A Retention times of daidzein, genistein and biochanin A Daidzein and genistein contents of soybeans Genistein and biochanin A contents of chickpeas Daidzein, genistein and biochanin A contents of food materials...35 A.1 Peak areas of the analytes in standard mixture xii LIST OF FIGURES FIGURES 1.1 The relationship between various groups of phytoestrogens and members of each group The structural similarities of phytoestrogens to oestradiol Isoflavone aglucones: daidzein, formononetin, genistein, biochanin A and glycitein Outline of the experimental procedure Chromatogram of the standard mixture Spectra of (a)daidzein, (b)genistein, (c)biochanin A Chromatograms of (a)haricot bean, (b)chickpea 23 Chromatograms of (a)green lentil, (b)red lentil, (c)soybean (cont d) 24 Chromatograms of (a)licorice root, (b)yarrow, (c)chestnut (cont d)..25 Chromatograms of (a)prunes, (b)raisins, (c)currants (cont d) 26 Chromatograms of (a)black cumin, (b)dried apricot, (c)dried parsley (cont d).27 Chromatograms of (a)dried dates, (b)dried figs, (c)sage (Aegean) Chromatograms of (a)sage(mediterranean), (b)grapevine, (c)gilaburu Overlaid (a)chromatograms of soybean and standard mixture, (b)spectra of daidzein and peak (8.668min), (c)spectra of genistein and peak (11.764min) Overlaid (a)chromatograms of chickpea and standard mixture, (b)spectra of genistein and peak(12.44min), (c)spectra of genistein and peak (2.327min) Chromatograms of (a)genistein and biochanin A standard mixture, (b)chickpea, (c)chickpea (black) laid over chickpea spiked with genistein and biochanin A standards (blue) xiii LIST OF ABBREVIATIONS RP-HPLC UV DAD ER HRT GC LC MS Reversed-Phase High-Performance Liquid Chromatography Ultra-Violet Detector Diode-Array Detector Estrogen Receptor Hormone Replacement Therapy Gas Chromatography Liquid Chromatography Mass Spectrometry xiv CHAPTER 1 INTRODUCTION 1.1. Phytoestrogens Phytoestrogens are naturally occurring chemicals of plant origin that have the ability to cause estrogenic and/or anti-estrogenic effects due to their structural similarities to the human hormone oestradiol (17-estradiol). (Setchell and Cassidy, 1999) Classes of Phytoestrogens Phytoestrogens Flavonoids Non-flavonoids Isoflavones Coumestans Prenyl flavonoids Lignans Genistein Coumestrol 8-Prenylnaringenin Lariciresinol Daidzein 6-Prenylnaringenin Isolariciresinol (Equol) Xanthohumol Matairesinol Glycitein Isoxanthohumol Secoisolariciresinol Biochanin A (Enterodiol) Formononetin (Enterolactone) Figure 1.1 The relationship between various groups of phytoestrogens (given in bold) and members of each group. (The compounds in paranthesis are not inherently present in plants but are oestrogenic products resulting from metabolism of members of that class of phytoestrogens.) 1 The majority of phytoestrogens belong to a large group of substituted phenolic compounds known as flavonoids. There are several groups of flavonoids with estrogenic properties. Of these, coumestans and isoflavones possess the greatest estrogenic activity (Collins et. al., 1997). A class of prenylated flavonoids with estrogenic activities intermediate to those of the coumestans and isoflavones has recently been identified (Milligan et. al., 1999). Lignans, a class of non-flavonoid phytoestrogens, have also been shown to exert estrogenic effects (Setchell and Adlercreutz, 1988). The relationship between these types of phytoestrogens and the names of the compounds most commonly found in food from these four groups are summarized in Figure Structural Similarities of Phytoestrogens to Oestradiol Similarity of phytoestrogens to estrogens at the molecular level provides them the ability to mildly mimic and in some cases act as an antagonist to estrogen (Oomah, 22). Common features of phytoestrogens and oestradiol are listed in Table 1.1 (Mazur and Adlercreutz, 2). Table 1.1. Key structural elements crucial for estradiol-like action - Presence of the phenolic ring indispensable for binding to estrogen receptors (ERs) - Role of the ring of isoflavones mimicking the ring of estrogens at receptor binding - Low molecular weights, similar to that of estradiol (C 18 H 24 O 2 ) (MW = 272) - Distance between two aromatic hydroxyl groups in the nucleus of the isoflavones almost identical to the distance between two hydroxyl groups of estradiol - Optimal pattern of hydroxylation, i.e., hydroxyl substituents at 4, 5, and 7 positions (e.g., genistein) The structural similarities between members of the four main groups of phytoestrogens identified in Figure 1.1 and oestradiol are shown in Figure 1.2 (Food Standards Agency, 23) 2 Figure 1.2 The structural similarities of phytoestrogens to oestradiol. (The similarity of the structure of oestradiol and examples from the four classes of phytoestrogens from Figure 1.1. All the structures possess the phenolic (A) and hydroxyl (B) moieties outlined in boxes on the oestradiol structure and the distances between the two groups in each compound are similar.) 3 Oestrogenic Properties of Phytoestrogens In 194s, it was first realized that some plant-derived compounds could cause estrogenic effects in animals (Bennetts et. al., 1946). Sheep grazing on pastures containing red clover had multiple fertility problems. The clover in these pastures had high amounts of the isoflavones, formononetin and biochanin A (Rossiter and Beck, 1966). The phytoestrogens, daidzein and genistein were responsible for the infertility of some captive cheetahs fed a soybean enriched diet subsequently found to contain high quantities of these compounds (Setchell et al., 1987). Evidence is beginning to accrue that phytoestrogens may begin to offer protection against a wide range of human conditions, including breast, bowel, prostate, and other cancers; cardiovascular disease; brain function; alcohol abuse; osteoporosis; and menopausal symptoms (Bingham et. al., 1998). The basis for these effects has not been established, but the weak estrogenic activity of isoflavones may be a factor in conferring these properties (Oomah, 22). The incidence of a number of cancers, including those of the breast and prostate, has been found to be much higher in Western populations compared with that in countries such as Japan and China. Epidemiological and migrant studies have suggested that racial characteristics and other factors including lifestyle, diet and fat or fibre intake may play a role in the aetiology of these diseases. One notable dietary difference is the relatively high consumption of soy and soy-based foods amongst Asian populations. Comparison of estimated dietary isoflavone intakes in Western and Eastern (e.g. Japanese and China) populations illustrate that Eastern populations have a significantly higher intake of phytoestrogens. Estimates suggest that the average Japanese consumer is exposed to approximately 25-1 mg isoflavones/day, while an average United Kingdom consumer ingests approximately 1 mg isoflavones/day (Food Standards Agency, 23). As such; soy, which has been known to be the richest source of isoflavones, has attracted much attention as a potential chemoprotective factor (Bingham et al., 1998; Cassidy and Faughnan, 2). 4 Oestrogenic Potencies of Phytoestrogens In general, phytoestrogens are relatively weak oestrogens, requiring much higher concentrations than oestradiol to produce an equivalent biological response. Since potency values can vary significantly between methods, relative absolute estrogenic potency of phytoestrogens is difficult to determine. However, taking the results of both in vitro and in vivo studies together, a single rank order of oestrogenic potency of phytoestrogens may be estimated: oestradiol coumestrol genistein, equol glycitein 8-prenylnaringenin daidzein formononetin, biochanin A, 6- prenylnaringenin, xanthohumol, isoxanthohumol. (Food Standards Agency, 23) Since coumestans, being reported to be the most potent phytoestrogen (Pelissero et. al., 1991), are found predominantly in clover and alfalfa plants (Miksicek, 1993) and so are rare components of the human diet (Adlercreutz, 1997); isoflavones attract great deal of interest in today s studies due to wider range of foods containing them Isoflavones Isoflavones are polyphenolic phytoestrogens that occur mainly as gluco-conjugates (glucosides) of genistein, daidzein, and glycitein (Clarke et al., 22). They enjoy a restricted distribution in the plant kingdom and are predominantly found in leguminous plants (Dixon and Ferreira, 22). The main dietary sources of isoflavones are soybeans and soyfoods (Lapcik et. al., 1998) Classes of Isoflavones The most prevalent isoflavones present in plant-based foods are as follows (Bingham et al., 1998) (Figure 1.3) : genistein daidzein glycitein biochanin A (methylated derivative of genistein) formononetin (methylated derivative of daidzein) 5 Figure 1.3 Isoflavone aglucones: daidzein, formononetin, genistein, biochanin A and glycitein Physical and Chemical Properties of Isoflavones Water Solubility Isoflavones are low molecular weight (MW of daidzein = 254, MW of genistein = 27, MW of biochanin A = 284) hydrophobic compounds. The aqueous solubilities of the isoflavone aglucones are low and due to the acidic nature of the phenolic groups are ph dependent. The methylated derivatives, biochanin A and formononetin are less soluble than genistein and daidzein, respectively. (Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment, 2) Conjugation to glucose, glucuronide or sulphate groups increases the solubility (Setchell and Cassidy, 1999). 6 Chemical Stability The isoflavone aglucones are stable under physiological conditions. Under acidic conditions, the glucosides can be deconjugated to give aglucones. In the body, enzymes in the gut and liver can carry out these reactions during metabolism. (Food Standards Agency, 23) Absorption, Distribution, Metabolism and Excretion of Isoflavones In plants, isoflavones are present as glucosides. Processing reduces the isoflavone content and can partially convert them to aglucones. So, isoflavones are ingested mainly as glucosides and undergo hydrolysis by gut bacterial and mammalian enzymes prior to absorption. (Wang et. al., 22) Following absorption of the aglucones, these compounds are reconjugated with sulphate and glucuronide and excreted in the bile or urine (Bingham et al., 1998). Gut microflora can also modify estrogenic isoflavones into more active forms. The methylated isoflavones, formononetin and biochanin A are demethylated by gut microflora to daidzein and genistein, respectively Analysis of Phytoestrogens Isolation of Phytoestrogens Isoflavones are often present as glucosides in plants. Since acidic conditions make glucosides to deconjugate into aglucones, most extraction procedures involve acid hydrolysis. Hydrolysis with 1-2 M hydrochloric acid at 1 ºC or refluxing with acid in the presence of ethanol has been used to form the aglucones (Wilkinson et. al., 22). However, there are some reports indicating genistein to be unstable under acid hydrolysis conditions (Franke et. al., 1994; Garrett et. al., 1999). For analysis of soy foods, typical extraction conditions that have been used are stirring-freeze-dried powdered samples with methanol-water (8:2, v/v) at room temperature or 4 ºC, or with a mixture of acetonitrile - hydrochloric acid (.1 M) - 7 water (Wang and Murphy, 1994; Murphy et. al., 1997; Song et. al., 1998; Murphy et. al., 1999; Griffith and Collison, 21). Using acidified solvents has
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